1,3-Butadiene (hereinafter butadiene) is an important monomer for the production of synthetic rubbers including styrene-butadiene-rubber (SBR), polybutadiene (PB), styrene-butadiene latex (SBL), acrylonitrile-butadiene-styrene resins (ABS), nitrile rubber, and adiponitrile, which is used in the manufacture of Nylon-66 (White, Chemico-Biological Interactions, 2007, 166, 10-14).
Butadiene is typically produced as a co-product from the steam cracking process, distilled to a crude butadiene stream, and purified via extractive distillation (White, Chemico-Biological Interactions, 2007, 166, 10-14).
On-purpose butadiene has been prepared among other methods by dehydrogenation of n-butane and n-butene (Houdry process); and oxidative dehydrogenation of n-butene (Oxo-D or O-X-D process) (White, Chemico-Biological Interactions, 2007, 166, 10-14).
Industrially, 95% of global butadiene production is undertaken via the steam cracking process using petrochemical-based feedstocks such as naphtha. Production of on-purpose butadiene is not significant, given the high cost of production and low process yield (White, Chemico-Biological Interactions, 2007, 166, 10-14).
Given a reliance on petrochemical feedstocks and, for on-purpose butadiene, energy intensive catalytic steps; biotechnology offers an alternative approach via biocatalysis. Biocatalysis is the use of biological catalysts, such as enzymes, to perform biochemical transformations of organic compounds.
Accordingly, against this background, it is clear that there is a need for sustainable methods for producing intermediates, in particular butadiene, wherein the methods are biocatalyst based (Jang et al., Biotechnology & Bioengineering, 2012, 109(10), 2437-2459).
Both bioderived feedstocks and petrochemical feedstocks are viable starting materials for the biocatalysis processes.
The generation of two vinyl groups into medium carbon chain length enzyme substrates is a key consideration in synthesizing butadiene via biocatalysis processes.
There are no known enzyme pathways leading to the synthesis of butadiene in prokaryotes or eukaryotes. Three potential pathways have been suggested for producing 1,3-butadiene from biomass-sugar: (1) from acetyl-CoA via crotonyl-CoA; (2) from erythrose-4-phosphate; and (3) via a condensation reaction with malonyl-CoA and acetyl-CoA. However, no information using these strategies has been reported (Jang et al., Biotechnology & Bioengineering, 2012, 109(10), 2437-2459).
The closest analogous compound synthesized by prokaryotes or eukaryotes is 2-methyl-1,3-butadiene (isoprene), given the short five carbon chain length and two vinyl groups. Isoprene may be synthesised via two routes leading to the precursor dimethylvinyl-PP, viz. the mevalonate and the non-mevalonate pathway (Kuzuyama, Biosci. Biotechnol. Biochem., 2002, 66(8), 1619-1627).
The mevalonate pathway incorporates a decarboxylase enzyme, mevalonate diphosphate decarboxylase (hereafter MDD), that generates the first vinyl-group in the precursors leading to isoprene (Kuzuyama, Biosci. Biotechnol. Biochem., 2002, 66(8), 1619-1627).
Mevalonate diphosphate decarboxylase (EC 4.1.1.33) may thus be earmarked as a candidate enzyme in the synthesis of butadiene from non-native substrates.
In elucidating the role of the 3-methyl group associated with the native substrate, mevalonate diphosphate, it has been demonstrated that the turn-over number, keat, for 3-hydroxy-5-diphosphatepentanoic acid as shown in FIG. 12(a) is dramatically lower at 0.23±0.05 [s−1] as opposed to the nominal 8.33±1 [s−1] for the native substrate (Dhe-Paganon et al., Biochemistry, 1994, 33, 13355-13362). In addition, the reaction with substrate only progressed as far as phosphorylation of the 3-hydroxyl group, i.e., no decarboxylated product was detectable, implying that the decarboxylation rate is decreased at least 300 fold compared to the native substrate. In conclusion, the 3-methyl group was deemed indispensible in stabilizing the carbo-cation transition state (Dhe-Paganon et al., Biochemistry, 1994, 33, 13355-13362).
It has been demonstrated that the MDD enzyme from Saccharomyces cerevisiae accepts 3-hydroxy-3-methyl-butyrate (FIG. 12(b)), which includes the 3-methyl group stabilizing the carbocation transition state, as a substrate converting the substrate to isobutene. However, the specific activity is dramatically lower at 4.8·10−6 [μmol/(min·mg)] as opposed to the native substrate activity of 6.4 [μmol/(min·mg)] (Gogerty & Bobik, Applied & Environmental Microbiology, 2010, 76(24), 8004-8010).
The key substrate binding interactions of serine and arginine residues on the periphery of the catalytic cleft with the pyrophosphate group of the native substrate mevalonate diphosphate have been elucidated. Correct substrate orientation within the catalytic cleft is thus important to enzyme activity, which plausibly accounts for the low activity of MDD when accepting 3-hydroxy-3-methyl-butyrate (FIG. 14(b)) as substrate (Barta et al., Biochemistry, 2012, 51, 5611-5621).
The importance of the 3-methyl group and the pyrophosphate group associated with the native substrate in underpinning the activity of MDD teaches against using MDD in the synthesis of butadiene from non-native precursors that do not contain these key groups.
The enzyme, isoprene synthase (hereinafter ISPS), generates the second vinyl group in the final precursor, dimethylvinyl-PP, of isoprene synthesis.
Isoprene synthase (EC 4.2.3.27) may thus be earmarked as a candidate enzyme in the synthesis of butadiene from non-native substrates.
Similar to MDD, the 3-methyl group associated with the native substrate dimethylvinyl-PP plays an important role in stabilizing the carbo-cation that has been postulated as a transient intermediate (Silver & Fall, J. Biol. Chem., 1995, 270(22), 13010-13016; Kuzma et al., Current Microbiology, 1995, 30, 97-103).
The importance of the 3-methyl group in underpinning the activity of ISPS teaches against using ISPS for the synthesis of butadiene from non-native precursors that do not contain the 3-methyl group.
In addition to MDD and ISPS, microorganisms can generate vinyl groups in metabolites typically via dehydratase, ammonia lyase, desaturase, or decarboxylase activity. However, these enzyme activities rarely catalyse the formation of terminal vinyl groups. Dehydratases and ammonia lyases typically accept fatty acid analogues that have activated hydrogen atoms or aromatic compounds, where the aromatic ring serves as an electron withdrawing group. Desaturases predominate in fatty acid synthesis, generating unsaturated bonds at fixed non-terminal positions along long chain fatty acids. In turn, decarboxylases acting on the terminal carboxyl group typically leave the associated alpha functional group at the terminal position after catalysis. Therefore, the associated enzymatic activity of these enzymes teaches against their use for the generation of terminal vinyl groups in short or medium chain carbon metabolites leading to the synthesis of butadiene.